Semaphorins are a family of membrane bound and soluble proteins classified into eight sub-classes based on their structural domains Semaphorins mainly regulate focal adhesion assembly/disassembly and induce cytoskeletal remodeling, thus affecting cell shape, cell attachment to the extracellular matrix, cell motility, and cell migration. Although originally identified as axon guidance factors that control the development of central nervous system, different Semaphorins have been shown to participate in many other processes, such as immune response, angiogenesis and lymphangiogenesis.
The seven class-3 Semaphorins (Sema3s), designated by the letters A-G, are the only vertebrate secreted Semaphorins. Neuropilins (Nrps) and the type A/D family Plexins (Plexin-A1, -A2, and -A3, and Plexin-D1) act as receptors for Sema3s. Each Sema3 family member shows distinct binding preference for Nrps. Each Sema3-Nrp complex associates with specific plexins to mediate downstream signaling.
Semaphorin 3C (Sema3C), a class-3 Semaphorin, is known to affect neuronal migration, such as by providing chemorepulsive cues to sympathetic neurons or chemoattractive cues to GABAergic neurons. In addition to its role as affecting neuronal migration, Sema3C has been shown to have additional functions. As opposed to most class-3 Semaphorins, found to function as inhibitors of endothelial cell migration and proliferation and as inhibitors of angiogenesis (Neufeld et al., 2008, Nat. Rev. Cancer, 8: 632-645), several studies indicate that Sema3C plays a distinct role in promoting angiogenesis, endothelial cell guidance and vascular morphogenesis. For example, Sema3C has been shown to induce proliferation and adhesion of mouse glomerular endothelial cells (MGEC). Additionally, Sema3C has been shown to induce an increase in MGEC directional migration and to stimulate MGEC capillary-like network formation on Collagen I gels (Banu N. et al., 2006, FASEB J., 20:2150-2152).
Sema3C has also been suggested to be involved in tumor progression, to promote tumor migration and to be highly expressed in metastatic tumor cells. For example, Sema3C was shown to be highly expressed in neoplastic cells of gastric cancer. Additionally, primary stomach tumors, as well as metastatic liver tumors, were significantly suppressed by Sema3C miRNA-induced silencing in nude mice (Miyato, H. et al., 2012, Cancer Sci., 103: 1961-1966).
The seven class-3 Semaphorins contain conserved furin like pro-protein convertases (FPPC) cleavage sites. The functional activity of class-3 Semaphorins is subject to regulation by cleavage at the FPPC cleavage sites. Furin like pro-protein convertases (FPPC) are a family of proteolytic enzymes which convert proteins from their inactive immature form to their active form through cleavage of the immature proteins at FPPC cleavage sites. Furin and six other members of this family (PC2, PC1/3, PACE4, PC4, PC5/6, and PC7) possess a strong preference for substrates containing the multi-basic cleavage motif Arg-X-Arg/Lys-Arg-X (Becker et al., 2010, J. Med. Chem., 53(3):1067-1075). Furin and its analogues are responsible for the maturation of a large number of inactive protein precursors and are thus involved in many normal physiological processes. Expression of FPPC is often upregulated in tumors and metastases (Seidah N. G. et al., 2012, Nat. Rev. Drug. Discov., 11:367-383).
In some cases, cleavage of class-3 Semaphorins at FPPC cleavage sites was found to result in the complete inhibition of biological activity, as in the case of Sema3B (Varshaysky et al., 2008, Cancer Res., 68:6922-6931). In another case, cleavage at FPPC cleavage sites of Sema3E was found to result in Sema3E's pro-metastatic activity. The proteolytic processing of Sema3E was found to have no role in regulating its inhibitory activity towards endothelial cells (Casazza et al., 2012, EMBO Mol. Med., 4:234-250).
Sema3C comprises conserved FPPC cleavage sites and further comprises a cleavage site for the ADAMTS1 extracellular metalloprotease. Naturally occurring Sema3C is present as a mixture of the FPPC-cleaved and non-cleaved forms. Cleavage of Semaphorin 3C induced by ADAMTS1 has been shown to promote in-vitro cell migration (Esselens C. et al., 2009, J. Biol. Chem., 285: 2463-2473). However, the functional properties of the various FPPC-cleaved and un-cleaved forms of Sema3C have not been characterized.
U.S. Patent Application Publication No. US2013/0028896 discloses methods, uses and pharmaceutical compositions for treatment of prostate cancer using a Sema 3C inhibitor that may be selected may from: an antibody, a Sema 3C peptide, an antisense RNA, a siRNA, a shRNA or a small molecule.
U.S. Patent Application Publication No. US2012/0101029 by some of the inventors of the present invention discloses a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pro-protein convertase resistant Semaphorin 3E.
Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients in developed countries. Although both forms of AMD (geographic atrophy or dry AMD and choroidal neovascularization or exudative AMD) affect central vision, the exudative form poses the greatest risk for severe visual loss. The exudative form is relatively fast progressing and is characterized by choroidal neovascularization (CNV), a process in which new leaky blood vessels originating in the choroid invade the retina. Among the angiogenic factors investigated, VEGF-A was found to be a key factor in animal models, and human wet AMD patients. Although additional angiogenic factors such as basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and hepatocyte growth factor (HGF) play a role as well. VEGF-A inhibitors such as ranibizumab (Lucentis™) or aflibercept (Eylea™) have proven to be effective in treating exudative AMD in the clinic and represent the current mainstay therapeutic treatment. The magnitude of non-response to treatment, defined as no improvement in visual acuity or in reading ability, is evident from recently published large clinical prospective randomized trials. In the Comparison of Age-Related Macular Degeneration Treatments Trial (CATT), more than 12% of patients lost more than 5 letters and more than 30% of patients experienced no significant change or suffered deterioration in visual acuity despite intensive and regular intra-vitreal injections of either bevacizumab or ranibizumab. Similarly, in the ANCHOR study, 22% of patients treated with monthly ranibizumab injections experienced no significant change or suffered deterioration in visual acuity Smaller prospective studies concluded that 45% of patients treated with intravitreal bevacizumab were non-responders. Furthermore, patients treated repetitively with anti-VEGF medication may be prone to develop geographic macular atrophy. There are also indications suggesting that patients develop resistance to treatment over time. Thus, novel therapeutics based on non-VEGF targeted mechanisms are required for a more effective treatment of this disease.